Name: positron emission tomography using 64-copper as a tracer for the study of copper-related disorders
Uploaded: 2023-04-28T12:40:00.000+00:00
Duration: 6 min 52 s
Description: Watch this Scientific Journal Video about Positron Emission Tomography Using 64-Copper as a Tracer for the Study of Copper-Related Disorders at JoVE.com

Supported by a grant from The Memorial Foundation of Manufacturer Vilhelm Pedersen &#38; Wife. The foundation played no role in the planning or any other phase of the study.

A few clinical trials using this 64Cu PET/CT or PET/MRI protocol have been approved by the Regional Ethics Committee of&#160;Region Midt, Denmark [1-10-72-196-16 (EudraCT 2016-001975-59), 1-10-72-41-19 (EudraCT 2019-000905-57), 1-10-72-343-20 (EudraCT 2020-005832-31), 1-10-72-25-21 (EudraCT 2021-000102-25), and 1-10-72-15-22 (EudraCT 2021-005464-21)]. Written informed consent was obtained from the participants at enrollment. The inclusion criteria for all participants were age &#62;18, and for females the use of safe contraception. The exclusion criteria for Wilson disease patients were decompensated cirrhosis, a Model for End-stage Liver Disease (MELD) score &#62;11, or a modified Nazer score &#62;6. The exclusion criteria for all participants were a known hypersensitivity to 64Cu or other ingredients in the tracer formula, pregnancy, breastfeeding, or a desire to become pregnant before the end of the trial.
1. Preparation of&#160;64CuCl2
<ol>
	<li>Dissolve solid 64CuCl2 in hydrochloric acid (0.1 M) and add sodium acetate buffer (0.5 M) to increase the pH to ~5. Formulate with saline and filter sterilize the solution by passing it through a 0.22 &#181;m filter (see Table of Materials). 
	NOTE: Sodium acetate buffer (0.5 M) is produced from sodium acetate trihydrate and sterile water that is passed through a 0.22 &#181;m sterilizing filter.</li>
	<li>For quality control of the produced 64CuCl2 solution, perform pH measurement, bacterial endotoxin testing, radiochemical purity determination, and radio nuclidic identification7,8.</li>
	<li>Store the product in a lead container at room temperature and keep it in quarantine until all quality control specifications have been satisfactorily met. 
	NOTE: For the present study, 64CuCl2 was produced with a radio nuclidic purity &#8805;99% and a radiochemical purity &#8805;95%. Solid 64CuCl2, used as starting material, was obtained from a commercial source (see Table of Materials).</li>
</ol>
2. Preparation of PET scanner
<ol>
	<li>Perform a quality check (QC)9 on the scanner, following the manufacturer&#39;s protocol (see Table of Materials). 
	NOTE: QCs must be performed daily in the morning before patient scans.</li>
</ol>
3. Drawing of tracer for intravenous (IV) injection and per oral (PO) administration
<ol>
	<li>Wear plastic gloves and remove the lid from the lead container.</li>
	<li>Use long tweezers to disinfect the rubber membrane of the tracer-containing glass bottle inside the lead container with a disinfection swab.</li>
	<li>Use tweezers to insert a short cannula (~0.5 mm x 16 mm) into the membrane to avoid spill-over from the vacuum inside the bottle.</li>
	<li>Use tweezers to insert a longer cannula to draw from. This cannula should be long enough to reach the bottom of the bottle (usually 50 mm).</li>
	<li>Ensure that the dose calibrator (see Table of Materials) is calibrated for 64Cu. Calculate an approximate volume to draw for the first draw. 
	NOTE: From the chemical quality control reports, the activity amount and volume of the liquid will be available, allowing for the calculation of an approximate volume to draw.</li>
	<li>Wear plastic gloves, insert an appropriately sized plastic syringe into the long cannula, and draw the calculated volume. This volume will depend on the concentration of 64Cu in the product and how much 64Cu is decided for the protocol (see Dose calculations under the representative results).</li>
	<li>Use tweezers to hold the cannula while moving the syringe to the dose calibrator to measure the radioactivity.</li>
	<li>Keep drawing until the appropriate radioactivity amount is reached. Approximately 5% of the tracer will remain in the syringe and cannula after injection. 
	NOTE: The 64Cu should not be diluted in salt water, as the tracer may precipitate. Thus, the syringe cannot be rinsed with saline water after the injection (this is not relevant for PO administration).</li>
	<li>With the tweezers, apply a cannula with a cap (~16 mm cannula) to close the syringe and store it in a lead container until application.</li>
</ol>
4. Application of the tracer
<ol>
	<li>IV injection
	<ol>
		<li>Insert an intravenous cannula (~22 G, 25 mm), preferably in a cubital vein, and rinse with saline water to ensure correct placement. 
		NOTE: A worksheet with the participant&#39;s name, a stamp or signature for tracer quality control release, and time points and radioactivity for drawing, injection, and leftover tracer should be available.</li>
		<li>Measure the radioactivity in the syringe using the dose calibrator available and note the time and activity on the worksheet.</li>
		<li>Transport the syringe in a lead container to the participant&#39;s bedside.</li>
		<li>If any spill-over occurs from the injection, place a napkin under the participant&#39;s elbow so the spilled radioactivity can be measured.</li>
		<li>With tweezers, remove the cap/cannula from the syringe, and with plastic gloves, connect the syringe to the IV access. Note the time on the worksheet and inject in one steady movement. 
		NOTE: As mentioned earlier, the syringe should not be rinsed with saline as the tracer may precipitate.</li>
		<li>Remove the syringe from the IV access, put on the cap/cannula, and place it in the lead container with the napkin if necessary.</li>
		<li>Rinse the IV access through with saline water.</li>
		<li>Note the time and leftover radioactivity in the syringe on the worksheet. 
		NOTE: The injected activity is calculated as the difference between the syringe activity before and after injection, but using the PET scan protocol to correct for decay. Thus, all three time points (draw, injection, and leftover measurements) and the measured radioactivity at the draw and leftover measurements are entered into the PET scan protocol when the participant is scanned (see step 5).</li>
		<li>Dispose of the leftover material appropriately, according to institutional safety regulations.</li>
		<li>Remove the IV access. In case any allergic reactions appear, leave the IV access in for 30 min.</li>
	</ol>
	</li>
	<li>Oral administration 
	NOTE: A worksheet with the participant&#39;s name, a stamp or signature for tracer quality control release, and time points and radioactivity for drawing, administration, and leftover tracer should be available.
	<ol>
		<li>In a disposable and soft plastic cup, pour around 100 mL of water or cordial; the 64Cu is tasteless. A disposable plastic straw and a small disposable plastic bag should be available.</li>
		<li>Measure the radioactivity in the syringe using the dose calibrator available and note the time and activity on the worksheet.</li>
		<li>Transport the syringe in a lead container to the participant&#39;s bedside. The participant should be seated in a bed or chair.</li>
		<li>Remove the cap/cannula from the syringe with tweezers and, wearing plastic gloves, inject the tracer into the cup, being careful not to spill any. Draw up a bit of the water/cordial and inject it into the cup again.</li>
		<li>Place a plastic straw in the cup (this is to minimize the risk of spill-over when the participant drinks).</li>
		<li>Note the time on the worksheet and let the participant drink. The cup should be as empty as possible.</li>
		<li>Put the empty cup and straw in the disposable plastic bag with the empty syringe and place them in the lead container.</li>
		<li>Note the time and measure the leftover radioactivity in the syringe. Note in the worksheet. 
		NOTE: The injected activity is calculated as the difference between the syringe activity before and after injection, but using the PET scan protocol to correct for decay. Thus, all three time points (draw, injection, and leftover measurements) and the measured radioactivity at the draw and leftover measurement are entered into the PET scan protocol when the participant is scanned (see Scan).</li>
		<li>Dispose of the leftover material appropriately, according to institutional safety regulations. 
		NOTE: Observing the participant for acute allergic reactions for 30 min after the intake may be appropriate.</li>
	</ol>
	</li>
</ol>
5. PET scans
<ol>
	<li>Place the participant in a supine position in the scanner.</li>
	<li>Perform overview CT or MR scanning to plan the specific region to be examined during the PET scan.</li>
	<li>Note the time of the draw, injection, and leftover measurement, and the radioactivity at the draw and leftover measurement in the PET protocol.</li>
	<li>Perform PET scanning following the steps below. 
	NOTE: The PET scan protocol must be standardized with regard to scan duration and image reconstruction parameters for all participants in the same study; published reports should be followed10,11,12.
	<ol>
		<li>Perform static PET scans with a scan time of 4.5 min/bed position for up to 24 h after tracer administration, and 10 min/bed position for up to 68 h after tracer administration (for further elaboration, see Scan under the representative results). 
		NOTE: During dynamic PET scanning, decay is continuously recorded and subsequently segmented into a frame structure. This allows for selection of frames from short time intervals to emphasize the dynamics of 64Cu distribution, and frames from longer time intervals to prioritize sensitivity. Typically, shorter intervals are selected right after injection and gradually increased thereafter10.</li>
	</ol>
	</li>
</ol>
6. Image reconstruction
<ol>
	<li>Reconstruct the images using the best available corrections for attenuation, scatter, time-of-flight, and point-spread function. 
	NOTE: The image reconstruction parameters must be carefully selected to optimize the image properties, such as signal recovery and signal-to-noise. For multicenter studies, it is critical to standardize the image quality between centers.</li>
</ol>
7. Data analysis
NOTE: The present study describes a simple method to quantify 64Cu content in the liver. The PET signal is measured as standard uptake value (SUV), the tissue radioactivity concentration adjusted for participant weight injected activity and/or kilobecquerel (kBq) per mL of tissue.
<ol>
	<li>Download data to a suitable program, for example, Dicom files, to PMOD. 
	NOTE: There are likely many different programs to analyze PET images, such as Hermes or PMOD (see Table of Materials).</li>
	<li>Adjust the CT/MR scan tones to differentiate the anatomical structures.</li>
	<li>Ensure that the anatomical scan and PET scan are overlapping.</li>
	<li>Working in the horizontal plane with the best MRI or CT scan, localize the liver and the big structures.</li>
	<li>Place an appropriate volume of interest (VOI) or multiple VOIs in the liver. 
	NOTE: A VOI is a defined area of tissue where the SUV is measured. A VOI consists of multiple regions of interest (ROIs), which are tissue areas in one plane. Many programs have spherical VOIs as a pre-setting, meaning multiple ROIs (one in each plane) do not have to be drawn to constitute a VOI. The right liver lobe tends to be more homogenous, and thus a good position to place VOIs.</li>
	<li>Place multiple VOIs in the right liver lobe in different horizontal planes to achieve the most precise measure of activity, as the SUV may vary somewhat (~5%) in the right liver lobe. Calculate the mean SUV of these VOIs.</li>
	<li>To quantify the SUV, for example, in the entire liver, draw ROIs covering the whole liver volume in each plane for dosimetry studies. 
	NOTE: Avoid big structures such as arteries and veins when using this method.</li>
</ol>

<ol>
	<li>T&#252;mer, Z., M&#248;ller, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Menkes+disease.">Menkes disease.</a> European Journal of Human Genetics. 18 (5), 511-518 (2010).</li><li>Ala, A., Walker, A. P., Ashkan, K., Dooley, J. S., Schilsky, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wilson's+disease.">Wilson's disease.</a> The Lancet. 369 (9559), 397-408 (2007).</li><li>Owen, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absorption+and+excretion+of+Cu64-labeled+copper+by+the+rat.">Absorption and excretion of Cu64-labeled copper by the rat.</a> The American Journal of Physiology-Legacy Content. 207 (6), 1203-1206 (1964).</li><li>Osborn, S. B., Roberts, C. N., Walshe, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake+of+radiocopper+by+the+liver.+A+study+of+patients+with+Wilson's+disease+and+various+control+groups.">Uptake of radiocopper by the liver. A study of patients with Wilson's disease and various control groups.</a> Clinical Science. 24, 13-22 (1963).</li><li>Vierling, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+radiocopper+into+ceruloplasmin+in+normal+subjects+and+in+patients+with+primary+biliary+cirrhosis+and+Wilson's+disease.">Incorporation of radiocopper into ceruloplasmin in normal subjects and in patients with primary biliary cirrhosis and Wilson's disease.</a> Gastroenterology. 74 (4), 652-660 (1978).</li><li>Gibbs, K., Walshe, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+with+radioactive+copper+(64Cu+and+67Cu);+the+incorporation+of+radioactive+copper+into+caeruloplasmin+in+Wilson's+disease+and+in+primary+biliary+cirrhosis.">Studies with radioactive copper (64Cu and 67Cu); the incorporation of radioactive copper into caeruloplasmin in Wilson's disease and in primary biliary cirrhosis.</a> Clinical Science. 41 (3), 189-202 (1971).</li><li>Kume, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+semi-automated+system+for+the+routine+production+of+copper-64.">A semi-automated system for the routine production of copper-64.</a> Applied Radiation and Isotopes: Including Data, Instrumentation and Methods for Use in Agriculture, Industry and Medicine. 70 (8), 1803-1806 (2012).</li><li>Ohya, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+preparation+of+high-quality+64Cu+for+routine+use.">Efficient preparation of high-quality 64Cu for routine use.</a> Nuclear Medicine and Biology. 43 (11), 685-691 (2016).</li><li>Koole, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EANM+guidelines+for+PET-CT+and+PET-MR+routine+quality+control.">EANM guidelines for PET-CT and PET-MR routine quality control.</a> Zeitschrift f&#252;r Medizinische Physik. , (2022).</li><li>Sandahl, T. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathophysiology+of+Wilson's+disease+visualized:+A+human+64Cu+PET+study.">The pathophysiology of Wilson's disease visualized: A human 64Cu PET study.</a> Hepatology. 76 (6), 1461-1470 (2022).</li><li>Munk, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+oral+zinc+regimens+on+human+hepatic+copper+content:+a+randomized+intervention+study.">Effect of oral zinc regimens on human hepatic copper content: a randomized intervention study.</a> Scientific Reports. 12 (1), 14714 (2022).</li><li>Kj&#230;rgaard, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravenous+and+oral+copper+kinetics,+biodistribution+and+dosimetry+in+healthy+humans+studied+by+64Cu]copper+PET/CT.">Intravenous and oral copper kinetics, biodistribution and dosimetry in healthy humans studied by 64Cu]copper PET/CT.</a> EJNMMI Radiopharmacy and Chemistry. 5 (1), 15 (2020).</li><li>Brewer, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zinc+acetate+for+the+treatment+of+Wilson's+disease.">Zinc acetate for the treatment of Wilson's disease.</a> Expert Opinion on Pharmacotherapy. 2 (9), 1473-1477 (2001).</li><li>Bush, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+copper+metabolism.+XVI.+Radioactive+copper+studies+in+normal+subjects+and+in+patients+with+hepatolenticular+degeneration.">Studies on copper metabolism. XVI. Radioactive copper studies in normal subjects and in patients with hepatolenticular degeneration.</a> Journal of Clinical Investigation. 34 (12), 1766-1778 (1955).</li><li>Murillo, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+value+of+64Cu+as+a+tool+to+evaluate+the+restoration+of+physiological+copper+excretion+after+gene+therapy+in+Wilson's+disease.">High value of 64Cu as a tool to evaluate the restoration of physiological copper excretion after gene therapy in Wilson's disease.</a> Molecular Therapy - Methods &amp; Clinical Development. 26, 98-106 (2022).</li><li>Squitti, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Copper+dyshomeostasis+in+Wilson+disease+and+Alzheimer's+disease+as+shown+by+serum+and+urine+copper+indicators.">Copper dyshomeostasis in Wilson disease and Alzheimer's disease as shown by serum and urine copper indicators.</a> Journal of Trace Elements in Medicine and Biology. 45, 181-188 (2018).</li></ol>

The authors have no conflicts of interest.

The method is like any other PET method, but the long half-life of 12.7 h offers the opportunity to investigate long-term copper fluxes (we have good results from up to 68 h after IV tracer injection). All steps in the protocol must be handled by personnel familiar with PET, although they are no more critical than any other PET examination.
Troubleshooting 
Because we often use 64Cu for long-term investigations, the PET signal will be noisier than usual. This i...

Copper is a vital catalytic cofactor that drives multiple important biochemical processes essential for life, and defects in copper homeostasis are directly responsible for human diseases. Mutations in the ATP7A or ATP7B genes, encoding copper-transporting ATPases, cause Menke&#39;s and Wilson diseases, respectively. Menke&#39;s disease (ATP7A) is a rare lethal disorder of intestinal copper hyperaccumulation with severe copper deficiency in peripheral tissues and deficits in copper-dependent enzymes1. Wilson disease (WD) (ATP7B) is a rare disease characterized by the inability to excrete excess copper to bile, resulting in copper overload and subsequent organ damage, most severely affecting the liver and brain2.
Studies on copper metabolism have utilized radiolabeled copper (usually 64-copper [64Cu] or 67-copper) for decades, and these studies have proven invaluable for our understanding of mammalian copper metabolism, including absorption site and excretion pathways3,4,5,6. Previously, gamma counters were used to detect the radioactive signal with a limited anatomical resolution, but recently, 64Cu positron emission tomography (PET) combined with computed tomography (CT) or magnetic resonance imaging (MRI) has been introduced in both human and animal studies. Today, PET scanners have such a high sensitivity that it is possible to track 64Cu for up to 70 h after injection. The long half-life of 12.7 h for 64Cu allows for the long-term assessment of copper fluxes. This improvement in resolution has just recently entered the field of copper studies, and studies on normal and pathological copper metabolism, as well as studies evaluating the impact of specific treatments, are starting to emerge. Additionally, the introduction of whole-body PET scanners with an extended field-of-view will further enhance the sensitivity of these examinations.
This methodological paper aims to enable clinicians and scientists to add 64Cu PET CT/MRI to the existing repertoire of tools as a robust and easy-to-use method for assessing copper metabolism in a manner comparable between nuclear medicine departments. The production of 64Cu copper can be carried out using different methods and is usually performed at special facilities. Among the nuclear reactions, the 64Ni (p, n) 64Cu method is widely used, since a high production yield of 64Cu can be obtained with low energy protons in this route7,8. A detailed description of the production methods is out of the scope of this work, and availability will differ by country and region.
In this article, we first describe the preparation of the necessary radiochemistry and the tracer. Then, the principles for preparing the PET/CT or PET/MRI scanners are demonstrated.

<table><tbody><tr><td>0.22 micrometer sterilizing filter</td><td>Merck Life Science</td><td></td><td></td></tr><tr><td>Cannula 21 G 50 mm</td><td>BD Microlance</td><td>301155</td><td></td></tr><tr><td>Cannula 25 G 16 mm</td><td>BD Microlance</td><td>300600</td><td></td></tr><tr><td>Dose calibrator</td><td>Capintec CRC-PC calibrator</td><td></td><td></td></tr><tr><td>PET/CT scanner</td><td>Siemens: Biograph</td><td></td><td></td></tr><tr><td>PET/MR scanner</td><td>GE Signa</td><td></td><td></td></tr><tr><td>PMOD version 4.0</td><td>PMOD Technologies LLC</td><td></td><td></td></tr><tr><td>Saline solution 0.9% NaCl</td><td>Fresenius Kabi</td><td></td><td></td></tr><tr><td>Sodium acetate trihydrate BioUltra</td><td>Sigma Aldrich</td><td>71188</td><td></td></tr><tr><td>Solid 64CuCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Danish Technical University Ris&amp;oslash;</td><td></td><td></td></tr><tr><td>Sterile water</td><td>Fresenius Kabi</td><td></td><td></td></tr><tr><td>Venflon 22 G 25 mm</td><td>BD Venflon Pro Safety</td><td>393280</td><td></td></tr></tbody></table>

positron emission tomography using 64-copper as a tracer for the study of copper-related disorders

Copper is an essential trace element, functioning in catalysis and signaling in biological systems. Radiolabeled copper has been used for decades in studying basic human and animal copper metabolism and copper-related disorders, such as Wilson disease (WD) and Menke&#39;s disease. A recent addition to this toolkit is 64-copper (64Cu) positron emission tomography (PET), combining the accurate anatomical imaging of modern computed tomography (CT) or magnetic resonance imaging (MRI) scanners with the biodistribution of the 64Cu PET tracer signal. This allows the in vivo tracking of copper fluxes and kinetics, thereby directly visualizing human and animal copper organ traffic and metabolism. Consequently, 64Cu PET is well-suited for evaluating clinical and preclinical treatment effects and has already demonstrated the ability to diagnose WD accurately. Furthermore, 64Cu PET/CT studies have proven valuable in other scientific areas like cancer and stroke research. The present article shows how to perform 64Cu PET/CT or PET/MR in humans. Procedures for 64Cu handling, patient preparation, and scanner setup are demonstrated here.

Dose calculation 
Based on dosimetry calculations, the effective radioactivity dose for IV administration is 62 &#177; 5 &#181;Sv/MBq tracer10. Thus, a 50 MBq dose is recommended depending on the time frame. Up to 75-80 MBq is applicable for longer examinations and provides good-quality images without exceeding an ethically approved dose. The effective dose for oral administration is 113 &#177; 1 &#181;Sv/MBq tracer, due to intestinal accumulation of the tracer. Thus, a lower...

Watch this Scientific Journal Video about Positron Emission Tomography Using 64-Copper as a Tracer for the Study of Copper-Related Disorders at JoVE.com

Positron Emission Tomography Using 64-Copper as a Tracer for the Study of Copper-Related Disorders

The present protocol describes how to perform 64Cu PET/CT and PET/MRI imaging in humans to study copper-related disorders, such as Wilson disease, and the treatment effect on copper metabolism.

Copper is an essential trace element, functioning in catalysis and signaling in biological systems. Radiolabeled copper has been used for decades in ...

positron-emission-tomography-using-64-copper-as-tracer-for-study

Research

JoVE Journal

Medicine

1.2K Views.  Aarhus University Hospital. The present protocol describes how to perform 64Cu PET/CT and PET/MRI imaging in humans to study copper-related disorders, such as Wilson disease, and the treatment effect on copper metabolism.

Video: Positron Emission Tomography Using 64-Copper as a Tracer for the Study of Copper-Related Disorders

A Basic Positron Emission Tomography System Constructed to Locate a Radioactive Source in a Bi-dimensional Space

A simple Positron Emission Tomography (PET) prototype has been constructed to fully characterize its basic working principles. The PET prototype was created by coupling plastic scintillator crystals to photomultipliers or PMT&#39;s which are placed at opposing positions to detect two gamma rays emitted from a radioactive source, of which is placed in the geometric center of the PET set-up. The prototype consists of four detectors placed geometrically in a 20 cm diameter circle, and a radioactive source in the center. By moving the radioactive source centimeters from the center the system one is able to detect the displacement by measuring the time of flight difference between any two PMT&#39;s and, with this information, the system can calculate the virtual position in a graphical interface. In this way, the prototype reproduces the main principles of a PET system. It is capable to determine the real position of the source with intervals of 4 cm in 2 lines of detection taking less than 2 min.

We present a simple but well-constructed Positron Emission Tomography (PET) system and elucidate its basic working principles. The goal of this protocol is to guide the user in constructing and testing a simple PET system.

A simple Positron Emission Tomography (PET) prototype has been constructed to fully characterize its basic working principles. The PET prototype was ...

Engineering

Investigations on the Ga(III) Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) complex and protocols for the preparation of its novel non-radioactive, i.e., natural Ga(III) as well as radioactive 68Ga complex. The ligand as well as the Ga(III) complex were characterized by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry and elemental analysis. 68Ga was obtained by a standard elution method from a 68Ge/68Ga generator. Experiments to evaluate the 68Ga-labeling efficiency of EOB-DTPA at pH 3.8&#8211;4.0 were performed. Established analysis techniques radio TLC (thin layer chromatography) and radio HPLC (high performance liquid chromatography) were used to determine the radiochemical purity of the tracer. As a first investigation of the 68Ga tracers&#39; lipophilicity the n-octanol/water distribution coefficient of 68Ga species present in a pH 7.4 solution was determined by an extraction method. In vitro stability measurements of the tracer in various media at physiological pH were performed, revealing different rates of decomposition.

Investigations on the GaIII Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

A procedure for the isolation of EOB-DTPA and subsequent complexation with natural Ga(III) and 68Ga is presented herein, as well as a thorough analysis of all compounds and investigations on labeling efficiency, in vitro stability and the n-octanol/water distribution coefficient of the radiolabeled complex.

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) ...

Chemistry

Harnessing the Bioorthogonal Inverse Electron Demand Diels-Alder Cycloaddition for Pretargeted PET Imaging

Due to their exquisite affinity and specificity, antibodies have become extremely promising vectors for the delivery of radioisotopes to cancer cells for PET imaging. However, the necessity of labeling antibodies with radionuclides with long physical half-lives often results in high background radiation dose rates to non-target tissues. In order to circumvent this issue, we have employed a pretargeted PET imaging strategy based on the inverse electron demand Diels-Alder cycloaddition reaction. The methodology decouples the antibody from the radioactivity and thus exploits the positive characteristics of antibodies, while eschewing their pharmacokinetic drawbacks. The system is composed of four steps: (1) the injection of a mAb-trans-cyclooctene (TCO) conjugate; (2) a localization time period during which the antibody accumulates in the tumor and clears from the blood; (3) the injection of the radiolabeled tetrazine; and (4) the in vivo click ligation of the components followed by the clearance of excess radioligand. In the example presented in the work at hand, a 64Cu-NOTA-labeled tetrazine radioligand and a trans-cyclooctene-conjugated humanized antibody (huA33) were successfully used to delineate SW1222 colorectal cancer tumors with high tumor-to-background contrast. Further, the pretargeting methodology produces high quality images at only a fraction of the radiation dose to non-target tissue created by radioimmunoconjugates directly labeled with 64Cu or 89Zr. Ultimately, the modularity of this protocol is one of its greatest assets, as the trans-cyclooctene moiety can be appended to any non-internalizing antibody, and the tetrazine can be attached to a wide variety of radioisotopes.

The bioorthogonal inverse electron demand Diels-Alder cycloaddition has been harnessed to create an effective and modular pretargeted PET imaging strategy for cancer. In this protocol, the steps of this methodology are described in the context of a model system employing the colorectal cancer targeted antibody huA33 and a 64Cu-labeled radioligand.

Due to their exquisite affinity and specificity, antibodies have become extremely promising vectors for the delivery of radioisotopes to cancer cells for ...

Bioengineering

Radiosynthesis and PET/CT Imaging of 68Ga-Labelled Nanobody in Xenograft Models

Radiosynthesis, Quality Control, and Small Animal Positron Emission Tomography Imaging of 68Ga-Labelled Nano Molecules

Small animal Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging techniques are crucial in preclinical cancer research, necessitating meticulous attention to radiotracer synthesis, quality assurance, and in vivo injection protocols. This study presents a comprehensive workflow tailored to enhance the robustness and reproducibility of small animal PET experiments. The synthesis process in the radiochemistry laboratory using 68Ga is detailed, highlighting stringent quality control and assurance protocols for each radiotracer production. Parameters such as concentration, molar activity, pH, and purity are rigorously monitored, aligning with standards applicable to human studies. This methodology introduces streamlined syringe preparation and a custom-designed 30G cannula for precise intravenous injections into mice. Monitoring of animal health during scanning, including temperature and heart rate, ensures their well-being throughout the procedure. Dosages for PET and SPECT scans are predetermined to balance data acquisition with minimizing radiation exposure to animals and researchers. Similarly, CT scans employ pre-programmed settings to limit radiation exposure, especially pertinent in long-term studies assessing treatment effects. By optimizing these steps, the workflow aims to standardize procedures, reduce variability, and enhance the quality of small animal PET/SPECT/CT imaging. This resource provides valuable insights for researchers seeking to improve the accuracy and reliability of preclinical investigations in molecular imaging, ultimately advancing the field.

This paper describes the radiosynthesis, formulation, quality control of a new radiolabeled probe (i.e., 68Ga-labeled nanobody NM-02), and its use for small animal PET/CT imaging in a xenograft model.

Small animal Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging techniques are crucial in preclinical ...

Cancer Research

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

Immunology and Infection

Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases

3-[18F]fluoro-4-aminopyridine, [18F]3F4AP, is a radiofluorinated analog of the FDA-approved drug for multiple sclerosis 4-aminopyridine (4AP). This compound is currently under investigation as a PET tracer for demyelination. We recently described a novel chemical reaction to produce metafluorinated pyridines consisting of direct fluorination of a pyridine N-oxide and the utilization of this reaction for the radiochemical synthesis of [18F]3F4AP. In this article, we demonstrate how to produce this tracer using an automated synthesizer and an in-house made flow hydrogenation reactor. We also show the standard quality control procedures performed before releasing the radiotracer for preclinical animal imaging studies. This semi-automated procedure may serve as the basis for future production of [18F]3F4AP for clinical studies.

Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases

We demonstrate the semi-automated radiochemical synthesis of [18F]3F4AP and quality control procedures.

3-[18F]fluoro-4-aminopyridine, [18F]3F4AP, is a radiofluorinated analog of the FDA-approved drug for multiple sclerosis 4-aminopyridine (4AP). This ...

Imaging CD19+ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography

Multiple sclerosis (MS) is the most common demyelinating central nervous system (CNS) disease affecting young adults, often resulting in neurological deficits and disability as the disease progresses. B lymphocytes play a complex and critical role in MS pathology and are the target of several therapeutics in clinical trials. Currently, there is no way to accurately select patients for specific anti-B cell therapies or to non-invasively quantify the effects of these treatments on B cell load in the CNS and peripheral organs. Positron emission tomography (PET) imaging has enormous potential to provide highly specific, quantitative information regarding the in vivo spatiotemporal distribution and burden of B cells in living subjects.
This paper reports methods to synthesize and employ a PET tracer specific for human CD19+ B cells in a well-established B cell-driven mouse model of MS, experimental autoimmune encephalomyelitis (EAE), which is induced with human recombinant myelin oligodendrocyte glycoprotein 1-125. Described here are optimized techniques to detect and quantify CD19+ B cells in the brain and spinal cord using in vivo PET imaging. Additionally, this paper reports streamlined methods for ex vivo gamma counting of disease-relevant organs, including bone marrow, spinal cord, and spleen, together with high-resolution autoradiography of CD19 tracer binding in CNS tissues.

Imaging CD19+ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography

This paper details methodology for radiolabeling a human-specific anti-CD19 monoclonal antibody and how to use it to quantify B cells in the central nervous system and peripheral tissues of a mouse model of multiple sclerosis using in vivo PET imaging, ex vivo gamma counting, and autoradiography approaches.

Multiple sclerosis (MS) is the most common demyelinating central nervous system (CNS) disease affecting young adults, often resulting in neurological ...

Neuroscience

PET-CT Scanning for Multi-Tracer Study of Cerebral Oxygen and Glucose Metabolism

Multi-Tracer Studies of Brain Oxygen and Glucose Metabolism Using a Time-of-Flight Positron Emission Tomography-Computed Tomography Scanner

The authors have developed a paradigm using positron emission tomography (PET) with multiple radiopharmaceutical tracers that combines measurements of cerebral metabolic rate of glucose (CMRGlc), cerebral metabolic rate of oxygen (CMRO2), cerebral blood flow (CBF), and cerebral blood volume (CBV), culminating in estimates of brain aerobic glycolysis (AG). These in vivo estimates of oxidative and non-oxidative glucose metabolism are pertinent to the study of the human brain in health and disease. The latest positron emission tomography-computed tomography (PET-CT) scanners provide time-of-flight (TOF) imaging and critical improvements in spatial resolution and reduction of artifacts. This has led to significantly improved imaging with lower radiotracer doses.
Optimized methods for the latest PET-CT scanners involve administering a sequence of inhaled 15O-labeled carbon monoxide (CO) and oxygen (O2), intravenous 15O-labeled water (H2O), and 18F-deoxyglucose (FDG)-all within 2-h or 3-h scan sessions that yield high-resolution, quantitative measurements of CMRGlc, CMRO2, CBF, CBV, and AG. This methods paper describes practical aspects of scanning designed for quantifying brain metabolism with tracer kinetic models and arterial blood samples and provides examples of imaging measurements of human brain metabolism.

Quantitative measurements of oxygen and glucose metabolism by PET are established technologies, but details of practical protocols are sparsely described in the literature. This paper presents a practical protocol successfully implemented on a state-of-the-art positron emission tomography-computed tomography scanner.

The authors have developed a paradigm using positron emission tomography (PET) with multiple radiopharmaceutical tracers that combines measurements of ...

Synthesis of 68Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /(T1)Magnetic Resonance Imaging

Here, we describe a microwave synthesis to obtain iron oxide nanoparticles core-doped with 68Ga. Microwave technology enables fast and reproducible synthetic procedures. In this case, starting from FeCl3 and citrate trisodium salt, iron oxide nanoparticles coated with citric acid are obtained in 10 min in the microwave. These nanoparticles present a small core size of 4.2 &#177; 1.1 nm and a hydrodynamic size of 7.5 &#177; 2.1 nm. Moreover, they have a high longitudinal relaxivity (r1) value of 11.9 mM-1&#183;s-1 and a modest transversal relaxivity value (r2) of 22.9 mM-1&#183;s-1, which results in a low r2/r1 ratio of 1.9. These values enable positive contrast generation in magnetic resonance imaging (MRI) instead of negative contrast, commonly used with iron oxide nanoparticles. In addition, if a 68GaCl3 elution from a 68Ge/68Ga generator is added to the starting materials, a nano-radiotracer doped with 68Ga is obtained. The product is obtained with a high radiolabeling yield (&#62; 90%), regardless of the initial activity used. Furthermore, a single purification step renders the nano-radiomaterial ready to be used in vivo.

Synthesis of 68Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /T1Magnetic Resonance Imaging

Here, we present a protocol to obtain 68Ga core-doped iron oxide nanoparticles via fast microwave-driven synthesis. The methodology renders PET/(T1)MRI nanoparticles with radiolabeling efficiencies higher than 90% and radiochemical purity of 99% in a 20-min synthesis.

Here, we describe a microwave synthesis to obtain iron oxide nanoparticles core-doped with 68Ga. Microwave technology enables fast and reproducible ...

Biology

Positron Emission Tomography

Positron emission tomography (PET) is a medical imaging technique involving radiopharmaceuticals &#8212; substances that emit short-lived radiation. Although the first PET scanner was introduced in 1961, it took 15 more years before radiopharmaceuticals were combined with the technique and revolutionized its potential.
One of the main requirements of a PET scan is a positron-emitting radioisotope, which is produced in a cyclotron and then attached to a substance used by the part of the body being investigated. This &#34;tagged&#34; compound, or radiotracer, is then put into the patient (injected via IV or breathed in as a gas), and how the tissue uses it reveals how that organ or other area of the body functions. For example, F-18 is produced by proton bombardment of 18O and incorporated into a glucose analog called fludeoxyglucose (FDG). How the body uses FDG provides critical diagnostic information. For example, since cancers use glucose differently than normal tissues, FDG can reveal cancers. The 18F emits positrons that interact with nearby electrons, producing a burst of gamma radiation. This energy is detected by the scanner and converted into a detailed, three-dimensional color image that shows how that part of the patient&#39;s body functions. Different levels of gamma radiation produce different amounts of brightness and colors in the image, which a radiologist can then interpret to reveal what is going on. For instance, a radioactive form of iodine can be used to monitor the thyroid and radioactive gallium can be used for cancer imaging.
The main advantage is that PET can illustrate the physiologic activity&#8212;including nutrient metabolism and blood flow&#8212;of the targeted organ or organs. In contrast, CT and MRI scans can only show static images. PET is widely used to diagnose a multitude of conditions, such as heart disease, the spread of cancer, certain forms of infection, brain abnormalities, bone disease, and thyroid disease. PET can also locate regions in the brain that become active when a person carries out specific activities, such as speaking, closing his or her eyes, etc. PET scans are now usually performed in conjunction with a computed tomography or magnetic resonance imaging scan for better data visualization and interpretation.
This content is derived from <a href="https://openstax.org/books/anatomy-and-physiology/pages/1-7-medical-imaging">Openstax, Anatomy and Physiology, Section 1.7: Medical Imaging</a> and <a href="https://openstax.org/books/chemistry-2e/pages/21-3-radioactive-decay">Openstax, Chemistry 2e, Section 21.3: Radioactive Decay</a> and <a href="https://openstax.org/books/physics/pages/22-5-medical-applications-of-radioactivity-diagnostic-imaging-and-radiation">Openstax, Physics, Section 22.5: Medical Applications of Radioactivity: Diagnostic Imaging and Radiation</a>

Positron emission tomography (PET) is a medical imaging technique involving radiopharmaceuticals &#8212; substances that emit short-lived radiation. ...

Positron Emission Tomography Using 64-Copper as a Tracer for the Study of Copper-Related Disorders

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Chapters in this video

A Basic Positron Emission Tomography System Constructed to Locate a Radioactive Source in a Bi-dimensional Space

Investigations on the Ga(III) Complex of EOB-DTPA and Its ⁶⁸Ga Radiolabeled Analogue

Harnessing the Bioorthogonal Inverse Electron Demand Diels-Alder Cycloaddition for Pretargeted PET Imaging

Radiosynthesis, Quality Control, and Small Animal Positron Emission Tomography Imaging of ⁶⁸Ga-Labelled Nano Molecules

Murine Lymphocyte Labeling by ⁶⁴Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

Automated Radiochemical Synthesis of [¹⁸F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases

Imaging CD19⁺ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography

Multi-Tracer Studies of Brain Oxygen and Glucose Metabolism Using a Time-of-Flight Positron Emission Tomography-Computed Tomography Scanner

Synthesis of ⁶⁸Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /(T₁)Magnetic Resonance Imaging

Positron Emission Tomography